The identification of disulfides in ricin D using proteolytic cleavage followed by negative-ion nano-electrospray ionization mass spectrometry of the peptide fragments.

Rationale: To use negative-ion nano-electrospray ionization mass spectrometry of peptides from the tryptic digest of ricin D, to provide sequence information; in particular, to identify disulfide position and connectivity.

Methods: Negative-ion fragmentations of peptides from the tryptic digest of ricin D was studied using a Waters QTOF2 mass spectrometer operating in MS and MS(2) modes.

Results: Twenty-three peptides were obtained following high-performance liquid chromatography and studied by negative-ion mass spectrometry covering 73% of the amino-acid residues of ricin D. Five disulfide-containing peptides were identified, three intermolecular and two intramolecular disulfide-containing peptides. The [M-H](-) anions of the intermolecular disulfides undergo facile cleavage of the disulfide units to produce fragment peptides. In negative-ion collision-induced dissociation (CID) these source-formed anions undergo backbone cleavages, which provide sequencing information. The two intramolecular disulfides were converted proteolytically into intermolecular disulfides, which were identified as outlined above.

Conclusions: The positions of the five disulfide groups in ricin D may be determined by characteristic negative-ion cleavage of the disulfide groups, while sequence information may be determined using the standard negative-ion backbone cleavages of the resulting cleaved peptides. Negative-ion mass spectrometry can also be used to provide partial sequencing information for other peptides (i.e. those not containing Cys) using the standard negative-ion backbone cleavages of these peptides.